Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a pivotal role in vertebrate intracellular signaling. They are a family of three very similar proteins that form tetrameric Ca2+ channels in endoplasmic reticulum (ER) membranes. For the last two decades I have been studying the molecular biology of IP3Rs and recently discovered that Bok, a very poorly understood Bcl-2 protein family member with unique features, binds very strongly to IP3Rs. The Bcl-2 protein family controls the intrinsic apoptosis pathway and is intensively investigated, both from a basic cell biology perspective, and as a target for anti-cancer drugs. Bok is most similar to the well-characterized pro-apoptotic proteins Bax and Bak, but its cellular role remains enigmatic and highly controversial; consensus is, however, that Bok is localized to the ER membrane and plays a role in apoptotic signaling. In the last 3 years, my laboratory has begun to characterize the novel Bok-IP3R interaction and its significance, but there is still much work to do. Now, through three Specific Aims, I plan to test the hypotheses that Bok binding to IP3Rs regulates IP3R function and is required for the role that Bok plays in apoptosis, leaning heavily on newly-developed cell lines in which Bok has been deleted using CRISPR/Cas9- based methods. Aim 1. Resolution of the binding site for Bok on IP3R1 and the development of binding-deficient IP3R1 constructs and peptides that inhibit the Bok-IP3R1 interaction. Results obtained will provide insight into how Bok interacts with IP3Rs and will generate tools useful for probing the significance of the Bok-IP3R interaction. Aim 2. Define whether Bok binding to IP3Rs regulates IP3R function. Results obtained should characterize a new mode of IP3R regulation and provide insight into the mechanism by which Bok influences apoptosis. Aim 3. Define the role of Bok in apoptotic signaling and the importance of the Bok-IP3R interaction to this role. Results obtained will be integrated with the data from the other Specific Aims to generate a model for how Bok influences apoptosis and will establish whether the Bok-IP3R interface is a drug target. In summary, this proposal represents the first effort at characterizing the nature and significance of the Bok-IP3R interaction and will undoubtedly shed light on IP3R function and the cellular role of Bok. The work will both advance our understanding of cell signaling and apoptosis, and may identify a new therapeutic target.